The homeodomain is a small structural motif in proteins that binds to specific target DNA sequences and thereby regulates gene transcription in eukaryotes. The homeodomain amino acid sequence determines the binding affinity and specificity for DNA. Thermal unfolding and temperature-induced changes in the secondary structure of the mutant H52R/NK-2 homeodomain protein (9713 MW) were investigated by differential scanning calorimetry (DSC), far-UV circular dichroism (CD), and intrinsic tryptophan fluorescence changes. In 50 mM Na- phosphate buffer, pH 7.4, the H52R/NK-2 protein reversibly unfolded with a transition temperature (Tm) of ca. 56 C as compared to a Tm value of ca. 39 C in 50 mM Hepes, pH 7.4 buffer. The stabilizing effect of the phosphate anion on the H52R/NK-2 homeodomain mutant protein may relate to DNA binding properties. Moreover, the unfolding was less cooperative in Hepes than in phosphate buffer, with overall unfolding enthalpies of ca. 34 and 40 kcal/mol, respectively. Adding 100 mM NaCl to buffers, improved refolding during heating and cooling cycles, as measured by CD. Good agreement was obtained between thermodynamic parameters obtained by DSC and temperature-induced CD changes at 222 nm. However, DSC data for the thermal unfolding of the H52R/NK-2 protein were fitted best to a non-two-state model, with ratios of calorimetric to vant Hoff enthalpy changes less than unity. Currently, H52R, H52R/W56T mutants and wild type protein interactions with short double-stranded DNA oligomers containing the specific target DNA sequence are being studied by isothermal titration calorimetry (ITC), DSC, and protein tryptophanyl fluorescence titrations. Association constants for the homeodomain protein-DNA interaction as well as protein conformational changes and stability changes that occur upon forming the homeodomain-DNA complex are being measured. - homeodomain proteins, DNA, thermal unfolding, stability, circular dichroism, calorimetry, fluorescence